Abnormal stem cell proliferation (numbers of divisions and clonal expansion) is likely a major risk factor and an early if not the earliest manifestation of intestinal tumorigenesis. However, stem cells are difficult to study since they are small in number and hard to identify among the more numerous terminally differentiated epithelial cells. This proposal will focus on the intestinal stem cells of mice deficient in DNA mismatch repair (MMR). The lack of MMR results in approximately 100-fold increases in mutation rates which increases the feasibility of two complementary approaches. First, the number of stem cell divisions can be estimated genetically from the accumulation of somatic mutations in microsatellite (MS) loci. Mutations can only accumulate in stem cells since all other cells are lost within days. Therefore, all epithelial cells essentially reflect the genotypes of the stem cells, which in turn should be a function of the numbers of their divisions. Preliminary data demonstrate that intestinal MS loci, as predicted by computer simulations, become progressively polymprphic with age. Second, frequencies of mutation and patterns of otherwise occult stem cell expansions will be deducted from the in situ loss of endogenous histologic markers (lacZ) due to mutation. The greater numbers of spontaneous mutations expected in the setting of deficient MMR should increase the efficiency of this well established histological technique. The numbers of stem cell divisions and their patterns of expansion will be observed in mice with germline mutations in Pms2, Mlh1, or Apc, and on diets (high fat/low calcium and calorie restricted) which increase or decrease the frequencies of neoplasia. These studies will determine whether currently occult alterations in stem cell division rates or in patterns of expansion are associated with intestinal neoplasia.